LOCUS       sequence-409105-        8090 bp    DNA     circular SYN 14-APR-2025
DEFINITION  P3-evoCjCas9-eAID and hU6-BsmBI-gRNA in AAV2 backbone (ITRs).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8090)
  AUTHORS   Schmidheini L, Mathis N, Marquart KF, Rothgangl T, Kissling L, Bock 
            D, Chanez C, Wang JP, Jinek M, Schwank G
  TITLE     Continuous directed evolution of a compact CjCas9 variant with broad
            PAM compatibility.
  JOURNAL   Nat Chem Biol. 2023 Sep 21. doi: 10.1038/s41589-023-01427-x.
  PUBMED    37735239
REFERENCE   2  (bases 1 to 8090)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2023 Sep 21. doi: 10.1038/s41589-023-01427-x."
COMMENT     Sequence Label: AAV-P3-evoCjCas9-eAID_gRNA
FEATURES             Location/Qualifiers
     source          1..8090
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     CDS             495..515
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4137..4385
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4428..4448
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4462..4686
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(4706..4778)
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     promoter        complement(4902..5150)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4960..4979)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5130..5150)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   5151..5280
                     /label=AAV2 ITR
     primer_bind     complement(5299..5316)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5299..5315)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5308..5330)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5457..5912
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5544..5563)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5754..5775
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5938..6042
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6043..6111,6112..6903)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6261..6280)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7074..7662
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7563..7582
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7950..7971
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7986..7991,7992..8009,8010..8016)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8024..8040
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     8029..8051
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8048..8064
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8048..8064
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct accggcgcgc cgggggaggc tgctggtgaa tattaaccaa ggtcacccca
      181 gttatcggag gagcaaacag gggctaagtc cacacgcgtg gtaccgtctg tctgcacatt
      241 tcgtagagcg agtgttccga tactctaatc tccctaggca aggttcatat ttgtgtaggt
      301 tacttattct ccttttgttg actaagtcaa taatcagaat cagcaggttt ggagtcagct
      361 tggcagggat cagcagcctg ggttggaagg agggggtata aaagcccctt caccaggaga
      421 agccgtcaca cagatccaca agctcctggg ccgccaccat gaaacggaca gccgacggaa
      481 gcgagttcga gtcaccaaag aagaagcgga aagtcagtga tagcctcttg atgaatagac
      541 gcgaattcct gtatcagttt aaaaacgtga gatgggcaaa aggccgacga gagacatatc
      601 tgtgctatgt cgttaagcgc agagattcag ccaccagttt ctctctcgac ttcggctacc
      661 tgcggaacaa gaatggttgc catgttgagc tcctgttcct gaggtatatc agcgactggg
      721 atttggaccc agggcggtgc tatagggtga catggtttat ttcctggtca ccttgttatg
      781 actgcgcgcg gcatgttgcc gattttctga gagggaaccc taacctgtct ctgaggatct
      841 tcaccgcgcg actgtacttc tgtgaggacc ggaaagccga acccgaggga ctgagacgcc
      901 tccacagagc gggtgtgcag attgccataa tgacctttaa ggactacttc tactgctgga
      961 acaccttcgt cgaaaatcac ggccggactt tcaaggcttg ggaaggattg catgaaaaca
     1021 gcgtcaggct ttccaggcag cttcgccgca ttcttctccc gtctggcgga tctagcggag
     1081 gatcctctgg cagcgagaca ccaggaacaa gcgagtcagc aacaccagag agcagtggcg
     1141 gcagcagcgg cgggtcagcc cgcatcctgg cctttgccat cggcatcagc agcatcggat
     1201 gggccttcag cgagaatgac gagctgaagg actgcggtgt gagaatcttt acaaaggtcg
     1261 agaaccccaa gaccggcgaa agcctggcgc tgccaagacg gctggcccgg tctgctcgga
     1321 agagatacgc ccggagaaaa gccagactga atcacctgaa acacctgatc gccaacgagt
     1381 tcaaactgaa ctacgaggac taccagagct tcgatgagag cttggctaag gcctacaaag
     1441 gcagcctgat cagcccctac gagctgagat tcagagctct gaatgagctc ctgagcaagc
     1501 aggacttcgc tagagtgatc ctgcacatcg ccaagcgaag aggctacgac gacatcaaga
     1561 actctgatga caaggagaag ggcgccattc tgaaagccat caagcagaac gaagagaaac
     1621 tggccaatta ccagagcgtg ggcgagtacc tgtacaagga gtacttccag aagttcaaag
     1681 aaaatagcaa agagttcacc aacgtgcgga acaagaagga gtcttatgaa agatgtatcg
     1741 cccagagctt cctgaaagac gagctgaaac tgatcttcaa gaagcaaaga gagtttggct
     1801 tcagcttcag caaaaaattt gaagaagagg tgctgtctgt cgccttctac aagagggctc
     1861 tgaaggactt cagccacctg gtgggcaatt gcagcttttt cacagacgaa aagcgggccc
     1921 ctaagaacag ccctctggcc ttcatgttcg tggctctgac cagaatcatc aacctgctga
     1981 acaacctgaa aaataccgag ggcatcctct ataccaagga cgatctgaac gccctgctga
     2041 atgaggtgct caagaatggc accctgacct acaagcaaac aaaaaaactg ctgggcctgt
     2101 ctgacgacta cgagtttaaa ggcgagaagg gcacctattt catcgaattt aagaagtaca
     2161 aggaattcat caaggcactg ggcgaacaca acctgtctca ggacgacctg aacgagatcg
     2221 ccaaggacat caccctgatc aaggatgaga tcaagctgaa aaaagctctg gccaagtacg
     2281 acctgaatca aaaccagatc gacagcttaa gcaagctgga atttaaggat cacctgaaca
     2341 tctcctttaa ggccctgaag ctggtgaccc cactgatgct ggaaggaaag aagtacgacg
     2401 aagcatgcaa cgagcttaac ctgaaggttg ctatcaacga ggataagaag gatttcctgc
     2461 ctgcctttaa cgagacatac tacaaggatg aggtgaccaa ccccgtggtg ctgagagcta
     2521 tcaaagagta cagaaaggtg ctgaacgccc tgctgaagaa gtacggcaag gtccacaaga
     2581 tcaatattga gctggcccgg gaggttggaa agaaccactc tcagagagca aagatcgaga
     2641 aggagcaaaa cgagaactac aaagcgaaga aggacgccga actggagtgc gagaagcttg
     2701 gcctgaagat caactctaag aatatcctga aactcagact tttcaaagaa cagaaggaat
     2761 tctgtgccta cagcggcgag aagatcaaaa tttctgacct gcaggatgaa aagatgctgg
     2821 agatcgacca catctaccct tactccagaa gcttcgacga cagctatatg aacaaagtgc
     2881 tggtgttcac aaagcagaac caggagaagc tgaatcagac ccctttcgag gccttcggca
     2941 atgactccgc caagtggcag aaaatcgagg tgctggccaa aaacctgcca accaagaaac
     3001 agaagagaat cctcgacaag aactacaagg acaaggaaca gaagaacttc aaggatcgga
     3061 acctgaacga cacccggtac atcgccaggc tggtgttaaa ttacaccaag gactacctgg
     3121 atttcctgcc cctgagcgac gacgagaaca ccaagctgaa cgacacacag aagggcagca
     3181 aggtgcacgt ggaagccaag agcggcatgc tgaccagcgc actgcgccac acttggggct
     3241 tcagcgctaa ggaccggaac aaccacctgc atcacgccat cgatgccgtg atcatagcct
     3301 acgccaacaa ctcaatcgtg aaagctttca gtgactttaa gaaagaacag gagagcaact
     3361 ctgccgaact gtacgccaag aaaattagcg agctggacta caagaacaaa cggaagttct
     3421 tcgaaccttt ctcaggattt agacagaagg tgctggataa gatcgatgaa atcttcgtgt
     3481 ccaagcccga gagaaagaag cctagcggag ccctgcacaa ggaaaccttc agaaaggaag
     3541 aagagttcta ccagtcttat gggggcaaag aaggcgtgct gaaggccctg gaactcggca
     3601 agatcagaaa ggtgaaggga aagattgtga agaacggcga catgttcaga gtggacatct
     3661 tcaagcacaa gaagaccaac aagttctatg ccgtgcctat ctacacaatg gatttcgccc
     3721 tgaaagtgct gcctaacaag gctgtcgcca gatctaagaa gggcgagatc aaggactgga
     3781 tcctgatgga cgaaaactac gagttctgct tcagcctgta caaggacagc ctgatcctga
     3841 tccaaacaaa gaagatgcag gagcctgaat tcgtgtacta caacgccttc acaagcagca
     3901 ccgtgagcct gatcgtgtct aaacatgata acaagttcga aaccctgtcc aagaaccaaa
     3961 agatcctgtt caagaacgcc aacgagaagg aagtgatcgc caagggcatc ggcattcaga
     4021 atctgaaggt gttcgagaaa tatatcgtgt ccgctctggg agaggttaca aaggccgagt
     4081 ttcggcagag agaagatttt aagaagagcg gcgggagcgg cgggagcggg gggagcacta
     4141 atctgagcga catcattgag aaggagactg ggaaacagct ggtcattcag gagtccatcc
     4201 tgatgctgcc tgaggaggtg gaggaagtga tcggcaacaa gccagagtct gacatcctgg
     4261 tgcacaccgc ctacgacgag tccacagatg agaatgtgat gctgctgacc tctgacgccc
     4321 ccgagtataa gccttgggcc ctggtcatcc aggattctaa cggcgagaat aagatcaaga
     4381 tgctgtctgg cggctcaaaa agaaccgccg acggcagcga attcgagccc aagaagaaga
     4441 ggaaagtcta atacatctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     4501 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     4561 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4621 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggtt
     4681 ctatggctcg agcggcccaa aaaaagcggt tttaggggat tgtaaccccg cagagtcccg
     4741 caaactcttt attctagtcc cttttcaggg actagaacag agacgtacaa aaaagagcaa
     4801 gaagctaaaa aagatttaaa aattattttt agcgcagtta atggaacagg aactaaattt
     4861 accccaaaat gtattttgtg tgctcattta caaccgtctc cggtgtttcg tcctttccac
     4921 aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc
     4981 attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac
     5041 gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttctaattat ctctctaaca
     5101 gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc aggaacccct
     5161 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     5221 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     5281 ccttaattaa cctaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     5341 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     5401 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc
     5461 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     5521 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5581 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5641 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     5701 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5761 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     5821 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5881 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     5941 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     6001 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     6061 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     6121 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     6181 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     6241 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     6301 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     6361 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6421 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6481 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6541 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6601 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6661 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6721 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6781 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6841 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6901 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6961 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     7021 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     7081 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     7141 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     7201 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     7261 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     7321 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     7381 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7441 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7501 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7561 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7621 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7681 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     7741 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     7801 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     7861 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     7921 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     7981 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     8041 tttcacacag gaaacagcta tgaccatgat tacgccagat ttaattaagg
//
